It has been shown for retroviral, influenza, and herpes viral glycoproteins that removal of N-linked glycans dramatically reduces immune reactivity (Alexander and Elder, 1984, Science, 226:1328-1330; Sjobloom et al., 1987, J. Gen. Virol. 68:549-554; Olofsson et al., 1990, J. Virol. 71:889-895). Considerable evidence has also accumulated suggesting that N-linked glycosylation is necessary for proper immunoreactivity or immunogenicity of human immuno-deficiency virus (HIV) gp120. For example, an extensive study comparing the immunogenicity of native glycosylated gp120 to that of env 2-3, a nonglycosylated form of the protein produced in yeast, was performed in baboons (Haigwood et al, 1992, J. Virol. 66:172-182). In this study, glycosylated gp120 induced high titers of neutralizing antibodies against the homologous and related viruses, and weak neutralizing titers against more distant viruses, while the nonglycosylated protein yielded only low neutralizing titers directed solely against the homologous virus. In addition, whereas the glycosylated protein was able to bind to CD4, the nonglycosylated protein was not. Other studies have shown that removal of N-linked glycans from native or recombinant gp120 reduces or abolishes binding activity of gp120 to CD4 and diminishes infectivity of HIV-1 (Matthews et al, 1987, PNAS 84:5424-5428; Fenouillet et al, 1989, J. Exp. Med. 169:807-821; Fenouillet et al, 1990, J. Virol. 64:2841-2848). Recent data show that removal of specific carbohydrates from recombinant gp160 reduced both its immunoreactivity with hyperimmune antisera and its immunogenicity in rabbits (Benjouad et al, 1992, J. Virol. 66:2473-2483). Finally, the epitopes of strongly neutralizing MAbs that have been isolated are destroyed by removal of N-linked glycans from the viral proteins (Fung et al, 1992, J. Virol. 66:848-856). These results demonstrate an important role for N-linked glycans in gp120 immunoreactivity and immunogenicity. These glycans may be acting either indirectly, by achieving the correct conformation of gp120, or directly by determining the formation of immunoreactive or immunogenic sites.
A major difficulty in the production of isolated gp120 domains is the fact that all of these domains are highly glycosylated. Considerable evidence suggests that glycans are needed either to achieve correct conformations or for the formation of the epitopes themselves.
The invention described here is intended to address this problem, but can also be used for the expression of non-gp120 glycopeptides whose immunogenicities or biological activities are dependent on correct glycosylation or conformation.